Tumor necrosis factor (TNF) signal transduction is involved in immune activation, cell death, and development. Specific intracellular signaling pathways induced by TNF include NF-kappaB activation, c-Jun N-terminal kinase (JNK) activation, and apoptosis. A number of other members of the TNF receptor family have related biological activities. The goal of this proposal is to identify essential components of TNF signal transduction using a genetic approach. By performing chemical mutagenesis in combination with NF-kappaB reporter selection, Jurkat T cell mutants will be generated which fail to activate NF-kappaB in response to TNF. These signaling mutants will be classified into complementation groups and tested for expression of gene products previously identified to play a role in TNF signaling (including but not limited to TNFR1, TRADD, TRAF2, TANK, RIP, and NIK). Expression cloning of cDNAs which reconstitute TNF signaling will be performed using repeated cycles of transient transfection, selection based on NF-kappaB reporter expression, and plasmid recovery. Individual cDNA clones identified by this approach will be characterized for their expression patterns, specificity of induction (i.e. requirements in TNF, IL-1, or TCR signaling), and specificity of effector function (i.e. NF-kappaB activation, JNK activation, or apoptosis induction). These mutants will allow unambiguous identification of gene products required for TNF induction of NF-kappaB and identify the functional role of these gene products relative to other TNF signal transduction intermediates.